Tyler D.
Jorgenson
*a,
Kashmeera D.
Baboolall
b,
Cristian
Suarez
c,
David R.
Kovar
cd,
Margaret L.
Gardel
*aefg and
Stuart J.
Rowan
*ah
aPritzker School of Molecular Engineering, University of Chicago, Chicago, IL, USA. E-mail: gardel@uchicago.edu; stuartrowan@uchicago.edu
bGraduate Program in Biosciences, University of Chicago, Chicago, IL, USA
cDepartment of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL, USA
dDepartment of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, USA
eInstitute for Biophysical Dynamics, University of Chicago, Chicago, IL, USA
fJames Franck Institute, University of Chicago, Chicago, IL, USA
gDepartment of Physics, University of Chicago, Chicago, IL, USA
hDepartment of Chemistry, University of Chicago, Chicago, IL, USA
First published on 2nd January 2024
In vitro studies of actin filament networks crosslinked with dynamic actin binding proteins provide critical insights into cytoskeletal mechanics as well as inspiration for new adaptive materials design. However, discontinuous variance in the physiochemical properties of actin binding proteins impedes holistic relationships between crosslinker molecular parameters, network structure, and mechanics. Bio-synthetic constructs composed of synthetic polymer backbones and actin binding motifs would enable crosslinkers with engineered physiochemical properties to directly target the desired structure–property relationships. As a proof of concept, bio-synthetic crosslinkers composed of highly flexible polyethylene glycol (PEG) polymers functionalized with the actin binding peptide LifeAct, are explored as actin crosslinkers. Using bulk rheology and fluorescence microscopy, these constructs are shown to modulate actin filament network structure and mechanics in a contour length dependent manner, while maintaining the stress-stiffening behavior inherent to actin filament networks. These results encourage the design of more diverse and complex peptide-polymer crosslinkers to interrogate and control semi-flexible polymer networks.
Point-mutations of existing ABPs or construction of chimeric ABPs allow for better control over their physical properties, e.g., length or binding strength, yielding further insights.15,16 However, difficulty in producing these systems prevents facile interrogation of the full physicochemical parameter space. Bio-synthetic crosslinkers provide an engineering handle through which the crosslinker's physiochemical properties can be independently and systematically tuned. Recent work by Lorenz et al. demonstrated that rigid DNA duplexes functionalized with either phalloidin or LifeAct actin binding peptides were able to form robust actin networks with properties akin to networks formed with biologically relevant ABPs.17 While these biomimetic crosslinkers represent a first step towards tunable actin crosslinkers, they are limited to rigid rod geometries given the relatively large persistence length, lp, of double stranded DNA (∼50 nm).18
Harnessing the expansive library of synthetic polymer backbones would enable a continuum of new bio-synthetic crosslinkers with well-defined physiochemical properties to be explored. However, it is an open question as to whether crosslinking constructs with synthetic polymer backbones will mimic their biopolymer counterparts as their persistence length is often significantly smaller than the polymer's contour length, lc, yielding unstructured globular crosslinkers (Fig. 1). Herein the utility of synthetic polymers as biomimetic crosslinker backbones via the development of LifeAct – polyethylene glycol (PEG) constructs is explored. These PEG-LifeAct constructs are demonstrated to be effective actin crosslinkers with contour length dependent network morphology and mechanics despite having equivalent hydrodynamic sizes. Additionally, the generated networks maintained many of the characteristic mechanical properties observed in native actin networks.
Fig. 1 Schematic comparison of the PEG-LifeAct constructs to that of typical biopolymer actin binding proteins and the chemical structure of the PEG-LifeAct construct's core. |
Name | Contour Length, lc (nm) | Spring Constant (pN μm−1) | Diameter (nm) |
---|---|---|---|
Contour length was calculated assuming a repeat distance of 0.28 nm and the number average molecular weight of the polymer. The spring constant was calculated assuming an entropic spring with the given contour length and a persistence length of 0.37 nm (see Note S1 for details). The diameter was measured via dynamic light scattering (see Fig. S4). | |||
PEG-2k-LA | 13 | 1300 | 4.7 ± 1.7 |
PEG-7k-LA | 45 | 370 | 6.2 ± 1.5 |
PEG-10k-LA | 64 | 260 | 5.6 ± 0.7 |
To test whether the synthesized PEG-LA constructs can restructure F-actin networks, fluorescently labeled G-actin was spontaneously assembled in the presence of the PEG-LA constructs and visualized via fluorescence microscopy. PEG-LA crosslinkers can restructure the F-actin network as can be seen in TIRF (top row) and confocal (bottom row) images presented in Fig. 2a. Bundling is observed for PEG-7k-LA and PEG-10k-LA at a relative concentration, Rc, of 33 mol% (top) and 15 mol% (bottom), while PEG-2k-LA shows no bundling. Time-lapse imaging of the bundling process suggests that G-actin is almost immediately organized into larger bundles of multiple filaments in the presence of PEG-7k-LA and PEG-10k-LA. The network morphology produced by either PEG-7k-LA or PEG-10k-LA features branched and curved bundles, reminiscent of network architectures generated by filamin.13 To quantify the differences in crosslinker bundling affinity, the percent of bundling, i.e., the fractional length of filaments contained within a bundle, was calculated from the TIRF images. The presented bundling affinities in Fig. 2b, suggest an increase in the PEG contour length improves the apparent bundling kinetics of the crosslinker despite having comparable hydrodynamic sizes.
The observed differences in crosslinker bundling ability and resulting actin network microstructures shown in Fig. 2a should translate into noticeable differences in network mechanics. To investigate such effects, the crosslinker dependent network mechanics were probed using bulk rheological measurements. Characteristic frequency sweeps of actin networks crosslinked with 15 mol% PEG-LA are shown in Fig. 3a. As may be intuited from microstructures presented in Fig. 2a, the storage modulus, G′, of actin networks crosslinked with PEG-10k-LA is higher than that of PEG-7k-LA and PEG-2k-LA networks at 15 mol%. In contrast to PEG-7k-LA and PEG-10k-LA, PEG-2k-LA crosslinked networks are more viscoelastic. To interrogate the impact of construct contour length on the linear mechanics of the actin networks, the loss factor, G′′/G′ = tan(δ), and the frequency dependence of G′ were analyzed as a function of Rc up to 20 mol% as shown in Fig. 3b. The tan(δ) (measured at 10 mHz) transitions from a value of ∼0.3 to ∼0.2 over the tested concentration regime for both PEG-2k-LA and PEG-7k-LA. Despite being the largest and most flexible crosslinker the loss factor remained below 0.2 for all PEG-10k-LA concentrations tested. Fitting the storage modulus from 0.01 to 1 Hz with a power law, G′(f) ∼ fS, reveals the moduli's frequency dependence (Fig. S8, ESI†). For PEG-10k-LA this steepness, S, is low for the entire tested concentration regime with an average value of 0.08. PEG-7k-LA networks have a similar steepness for Rc above 5 mol%. In contrast, PEG-2k-LA networks exhibit a much greater dependence between S and Rc. Only at a Rc of 20 mol% did PEG-2k-LA networks reach a value analogous to the longer PEG-LA crosslinked networks.
The lower tan(δ) and S for PEG-7k-LA and PEG-10k-LA compared to the PEG-2k-LA likely arises from differences in the bundling that is observed in Fig. 2. Bundled filaments have a higher bending modulus resulting in a more elastic network. Additionally, these results further support the observed differences in effective binding affinity suggested by the microstructure analysis presented in Fig. 2b. Elastic insensitivity to frequency occurs when the concentration of crosslinker is high enough to ensure filaments are always crosslinked, thus, arresting non-bending filament motion. This condition appears to be satisfied for concentrations of PEG-7k-LA and PEG-10k-LA above 4 mol% while higher concentrations of PEG-2k-LA are needed to reach such an arrested state. To better understand these observed differences for PEG-LA crosslinkers, the pseudo-plateau modulus, G0, of the crosslinked actin networks measured at 10 mHz was determined as a function of Rc. As shown in Fig. 4a, a plateau region exists until a critical concentration of crosslinker, , at which point G0 increases substantially in a log-linear manner for each crosslinker. The observed depends on the contour length of the crosslinker, with values of approximately 8, 5, and 2 mol% for PEG-2k-LA, PEG-7k-LA, and PEG-10k-LA, respectively (Fig. 4a). The lowering of with increasing contour length further confirms differences in effective crosslinking kinetics. The mechanical effectiveness of the crosslinkers, G0 ∼ Rxc, appears to vary inversely with the contour length of the construct. The scaling value, x, decreases from 1.5 ± 0.3 to 0.8 ± 0.1 for an increase in PEG contour length of 13 to 64 nm.
Interestingly, a similar relationship between crosslinker size and G0 ≈ Rxc scaling has been reported for semi-flexible DdFilamin-like biopolymer constructs composed of hisactophilin actin binding domains separated by a variable number of an IgG fold protein spacer.15 Specifically, a dimer of hisactophilin scaled with R1.2c while the largest construct studied scaled with R0.5c. The authors suggested the decrease in scaling of G0 with Rc was related to a reduced bundling propensity of the larger crosslinkers. Our results suggest that bundling propensity is not directly correlated with Rxc as the larger PEG-LA crosslinkers yield higher bundling affinity but lower elastic sensitivity to Rc. In a study of synthetic actin crosslinkers comprised of rigid 20 nm DNA duplexes functionalized with either phalloidin or LifeAct peptides, Lorenz et al. reported the phalloidin and LifeAct crosslinkers yield power law dependences of R0.4c and R1.1c, respectively.17 The authors concluded that the order of magnitude lower KD of phalloidin led to an increased partitioning of crosslinker into actin bundles leading to fewer elastically active inter-bundle crosslinks. The observed PEG-LA contour length dependent crosslinking kinetics may lead to a similar partitioning of crosslinker within the networks. Interestingly, when mechanical data presented in Fig. 4a are scaled by the low concentration G0 and , a master curve is generated with a scaling of 1.0 ± 0.1 (Fig. 4c). This result suggests that crosslinking kinetics plays a central role in determining a construct's mechanical effectiveness when all other crosslinker properties are constant.
While the scaling of G0 ≈ Rxc depends on the physiochemical properties of the crosslinker, the relationship between G0 and actin concentration, CA, has been shown to track closely with expected scaling laws derived from polymer physics.11,21–23 To determine if the expected scaling holds for the PEG-LA constructs, G0 was probed at a constant Rc of 15 mol% to ensure robust networks. The expected log-linear increase in G0 with increasing CA was observed for all crosslinkers (Fig. 4b). PEG-7k-LA and PEG-10k-LA networks scale with C2.3 ± 0.3A and C2.7 ± 0.2A, respectively (Fig. 4d). These observed values are within error of the expected scaling for a densely crosslinked (C2.5A) network when assuming a mechanical bending model of semi-flexible polymers.23 Surprisingly, the modulus of PEG-2k-LA networks is relatively insensitive to changes in actin concentration with scaling of G0 ∼ C1.4 ± 0.2A. While the limited data range used to determine the scaling warrants caution of over-analysis, the observed insensitivity to CA may arise from the previously discussed differences in linear network mechanics. Networks composed of PEG-2k-LA exhibit higher frequency dependence suggesting larger extents of filament mobility. For the tested range of CA and Rc of 15 mol%, the frequency dependence, S, is approximately 2× higher for PEG-2k-LA networks than the analogous PEG-7k-LA and PEG-10k-LA networks. The additional mobility in these networks indicates modes of stress dissipation other than the assumed bending modes that lead to the expected C2.5A scaling.
Collectively, the results presented in Fig. 2–4 demonstrate that highly flexible (lp ≪ lc) synthetic PEG constructs that have a lack of geometry can indeed act as effective actin crosslinkers with apparent contour length dependent control over network mechanics and structure. The observed contour length dependent network mechanics and structure can be attributed to the apparent crosslinking kinetic difference of the PEG-LA constructs. We posit that augmented crosslinking kinetics with increasing contour length results from improved positioning of the LifeAct binding domains. In aqueous conditions, PEG is well described as a freely jointed chain.24 As the LifeAct peptides are connected to the PEG chain ends, the average end-to-end distance, re, is a decent proxy for the LifeAct-LifeAct distance in the crosslinker. For PEG-2k-LA, the re is ∼50% of the hydrodynamic diameter suggesting a large fraction of constructs have the LifeAct functionalized chain-ends in close proximity, which inhibits effective crosslinking through the formation of singly bound constructs or doubly bound “loops”. In contrast, for PEG-7k-LA and PEG-10k-LA the re is ∼70% and 94% of the hydrodynamic diameter, respectively, suggesting the LifeAct domains are better positioned for inter-filament crosslinking (see Note S2 and Fig. S9, ESI†). The contour length dependent presented in Fig. 4a supports this physical view of PEG-2k-LA as higher concentrations are needed to overcome the fraction of constructs with inefficiently positioned LifeAct units. Moreover, these elastically ineffective bound PEG-2k-LA constructs would similarly lead to the observed inability of PEG-2k-LA to restructure the F-actin network's morphology and limit filament mobility.
Given how the extensible nature of PEG based constructs affected linear network mechanics, it was important to explore the impact on the actin network's non-linear mechanical response. A characteristic feature of actin networks is the pronounced stress-stiffening beyond a critical stress which represents a transition between bending to stretching dominated stress responses. Within this stiffening regime, the differential elastic modulus, K′, increases with applied pre-stress as K′ ≈ σ1.5.11 When networks crosslinked with the compliant ABP Filamin are stressed further, a scaling of unity with σ is observed, indicating a significant amount of stress is mediated through the crosslinker.25,26 To determine whether the highly compliant PEG-LA crosslinkers mediate stress rather than the actin filaments, K′ was measured as a function of applied pre-stress for networks with 15 mol% crosslinker at a variety of actin concentrations. As shown in Fig. 5, all tested PEG-LA networks stress-stiffened and could be collapsed by the zero-stress differential modulus, K0, and the critical stress, σc, to a master curve with a single stiffening regime. The scaling within this regime is well described by the expected power law σ1.5 suggesting the networks were still dominated by the stretching of the actin filaments rather than the extension of the PEG crosslinkers. Previously published coarse-grained simulations of two-dimensional actin networks suggested that crosslinker stiffnesses less than ∼100 pN μm−1 are needed for strain energy to be stored primarily in the crosslinker.27 Our experimental results bolster the validity of this mechanical threshold as the most compliant crosslinker tested herein has an approximate spring constant of ∼260 pN μm−1.
Collectively, the presented results show that crosslinkers composed of highly flexible synthetic polymer backbones (lp ≪ lc) have contour length dependent properties despite having indistinguishable hydrodynamic sizes. Moreover, the main impact of increasing contour length was an augmented effective crosslinking kinetics arising from improved geometric positioning of the LifeAct domains. It is hypothesized this improved positioning is a consequence of conformational statistics of an ideal chain. The increased crosslinking kinetics leads to improved bundling of filaments as well as more robust F-actin gels that behave analogously to native networks. For short contour length constructs, a larger fraction of conformations orient the LifeAct domains in close proximity resulting in the formation of elastically ineffective bound species. Such a phenomena would limit the construct's ability to crosslink filaments, hinder filament mobility, and reconstruct the network's structure. More broadly, the presented results highlight the potential of synthetic polymers as scaffolds for biomimetic actin crosslinkers. Future work could specifically tune polymer backbone flexibility to better probe the role of crosslinker stiffness on the network's stress-stiffening properties, as well as explore non-native geometries such as multi-armed crosslinkers or mixed binding domain systems. Such synthetic systems promise to provide greater insight into cytoskeletal functions as well as provide specific design rules for adaptive materials design.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d3sm01341c |
This journal is © The Royal Society of Chemistry 2024 |